成犬ならびに老犬の歯状回と海馬におけるグリア線維酸性蛋白免疫染色性の変化

誌名誌名 The journal of veterinary medical science

ISSNISSN 09167250

著者著者

Hwang, I.K.Choi, J.H.Li, H.ほか8名,

巻/号巻/号 70巻9号

掲載ページ掲載ページ p. 965-969

発行年月発行年月 2008年9月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

FULL PAPER Anatomy

Changes in Glial Fibrillary Acidic Protein Immunoreactivity in the Dentate Gyrus

and Hippocampus Proper of Adult and Aged Dogs

In Koo HWANG')， Jung Hoon CH0I2)， Hua U2)， Ki-Yeon Y002)， Dae Won KIM3)， Choong Hyun LEE')， Sun Shin YI')，

Je Kyung SEONG')， In Se LEE')， Yeo Sung YOON')* and Moo-Ho WON2，4)*

J)Department of Anatomy and Cell Biology， College ofVeterinary Medicine and BK21 Programfor Veterinary Sc附 lce，Seoul Natio叩 lUniversity， SeouI151-742， 2JDepartment of A叩 tomyand Neurobiology， College of Medicine， Hallym University， Chuncheon 200-702， JJCentral Research lnstitute， Natural F &P Coリ Ltd.，Chuncheon 200-160 and 4JlnstItute of Neurodegeneration and Neuroregeneration， Hallym University， Chuncheon 200-702， South Korea

(Receiv巴d10 January 2008/Accept巴d23 May 2008)

ABSTRA口 Astrocyt巴sperform neuron-supportive tasks， repair and scarring process in the central nervous system. ln this study， we observed glial fibrillary acidic protein (GFAP)， a marker for astrocytes， imrnunoreactivity in th巴dentategyrus and hippocampus proper (CAI-3 region) of adult (2-3 years of age) and aged (10-12 years of age) dogs. In the adult group， GFAP immunoreactive astrocytes wer巴distributedin all lay巴rsof the dentate gyrus and CAI-3 region， except in the stratum pyramidale of the CAI-3 region. In the aged group， GFAP immunoreactivity decreas巴dmarkedly in th巴 molecularlayer of the dentate gyrus. However， GFAP immunoreactivity in the CA 1-3 region incr巴as巴din a11 layers， and the cytoplasm of GFAP immunoreactive a山 ocyteswas hypertrophied. GFAP prot巴inlev els in the aged dentate gyrus decreased; however， GFAP levels in出eCAI-3 region increased. Thes巴resultssuggest that the morphology of as甘ocytesand GFAP prot巴inlevels in the hippocampal dentate gyrus and CA 1 region are changed， respectively， with age. KEY WORDS: aging， astrocyte， canine， glial fibrillary acidic protein， hippocampus.

Astrocytes， one of the neuroglia， perform neuron-sup-

portive tasks， including the biochemical support of endothe-

lial cells， which form the blood-brain barrier， the uptake of neurotransmitters from the synaptic cl巴ft，the release of energetic substrates that are essential to sustain high meta-

bolic activities at the synapse， and a principal role in repair

and scarring process in the brain [8]. Astrocytes ar巴charac-

terized by the expression of the intermediate filament， glial fibrillary acidic protein (GFAP) within their cytoplasm [4].

A well-known feature of reactive astrocytes increases the

production of intermediate filaments， and increases th巴

expression ofGFAP [21].

The hippocampus is a very important brain region to con-

trol cognitive functions [6]， and is the brain region most vul-nerable to aging processes [17， 18]. In addition， the hippocampus is one of the initial brain regions displaying

pathology and neuronal loss in Alzheimer's disease [26]，

The dog is widely accepted for aging study， because dogs

have the sameβamyloid sequenc巴 tohuman [12] and the

extent of s-amyloid has also been Iinked to cognitive dys-function in dogs [3， 10].

Although many studies on age-r巴latedchanges of astro-

cytes in the central nervous system in many sp巴ciesare con-

ducted [1， 2， 7， 9， 23， 25， 27]， few reports have demonstrated

* CORRESPONDENCE TO: Prof. YOON， Y. S.， Department of Anatomy and Cell Biology， College of Veterinary Medicine， Seoul National University， SeouI151-742， South Korea. 巴ーmail:ysyoon@snu.ac.kr Prof. WON， M.-H.， Department of Anatomy and Neurobiology， College of Medicine， Hallym University， Chuncheon 200-702， South Korea 巴-mail:mhwon@hallym.ac.kr

J. Vet. Med. Sci. 70(9): 965-969， 2008

the GFAP expression in the aged dog brain. In this study， therefore， we investigated age-related changes in GFAP

imrnunoreactivity and its protein levels in the hippocampus

to elucidate the correlation between ag巴andchange in astro-

cyte morphology.

MATERIALS AND METHODS

Experimental animals: The present study used the prog-

eny of German shepherd obtained from the special weapons

and tactics (SW A T)， South Korea. Male dogs were used at

2-3 years of age for adult group and 10-12 years of age for aged group. These animals did not show any clinical and

other signs in neural disorders. The procedures for handling

and caring for the animals adhered to the guidelines， which

are in compliance with the current intemational laws and

policies (NIH Guide for the Care and Use of Laboratory

Animals， NIH Publication No. 85-23， 1985， revised 1996)，

and they were approved by the Institutional Animal Care

and Use Commutee at Hallym's Medical Center. All of the

experiments were conducted to minimize the number of ani-

mals used and the suffering caused by the procedures used

in this study.

lmmunohistochemistry: For immunohistochemical analy-

sis， adult and aged dogs (n=5 at each age) were anesthetized with intravenous injection ofO.15 mg/kg ketamine (Yuhan， Seoul， South Korea) and 0.05 mg/kg xylazine (Bayer Ani-

mal Health， Suwon， South Korea) and perfused transcar-

dially with 0.1 M phosphate-buffered saline (PBS， pH 7.4) follow巴dby 4% paraformaldehyde in 0.1 M phosphate-

buffer (PB， pH 7.4)， The tissue processing and immunohis-tochemical staining was conducted by the methods of our

966 I.K目 HWANGET AL.

previous study [11]. In brief， tissues were serially sectioned into 30-μm coronal sections， and they were stained with diluted rabbit anti-GFAP antibody (1: 1 ，000， Chemicon，

Temecula， CA， U.S.A.) and subsequently exposed to bioti-nylated goat anti・rabbitIgG (1:200， Vector， Burlingame，

CA， U.S.A.) and streptavidin peroxidase complex (1 :200， Vector). They were then visualized by staining with 3，3¥ diaminob巴nzidinein 0.1 M Tris-HCl buffer (pH 7.2).

Western blot analysis: To confirm chang巴sin GFAP 1巴v-els in the hippocampus of adult and aged dogs， 2 animals in each group were sacrificed and used for Western blot analy-sis. After sacrificing them and removing the brain， th巴hip-pocampi were dissected with a surgical blade. The tissu巴swere homogenized in 50 mM PBS (pH 7.4) containing 0.1 mM ethylene glycol bis (2-aminoethyl Ether)-N，N，N' ，N' tetraacetic acid (pH 8.0)， 0.2% Nonidet P-40， 10 mM ethyl-endiamine tetraacetic acid (EDTA) (pH 8.0)，15 mM sodium pyrophosphate， 100 mM s-glycerophosph蹴，印刷 NaF，150 mM NaCI， 2 mM sodium orthovanadate， 1 m恥1phenyl-methylsulfonyl fluoride and 1 mM dithiothreitol (DTT). After centrifugation， the protein level was deterrnined in the supernatants using a Micro BCA protein assay kit with bovine serum albumin as the standard (Pierce Chemical， Rockford， IL， U.S.Aふ Aliquotscontaining 50μg of total protein were boiled in loading buffer containing 150 mM Tris (pH 6.8)， 3 mM DTT， 6% SDS， 0.3% bromophenol blue and 30% glycerol. The aliquots were then loaded onto a 10% polyacrylamide gel. After electrophoresis，出巴 g巴Iswere transferred to nitrocellulose transfer membranes (Pall Crop， East Hills， NY， U.S.A.). To reduce background stain-ing， th巴 membran巴swere incubated with 5% non-fat dry milk in PBS containing 0.1 % Tween 20 for 45 min， fol-lowed by incubation with rabbit anti-GFAP antiserum

(1: 1，000)， peroxidase-conjugated goat anti-rabbit IgG (Sigma， St. Louis， MO， U.S.A.) and an ECL kit (Pierce Chernical).

Quantification of data: Thirty sections per animal were selected in order to quantitatively analyze the relative opt卜

cal density (ROD) of GFAP immunoreactive structures in the hippocampus. The studied tissue sections were selected according to anatomical landmarks corresponding to the Figs. 54 and 55 of the Lim et al. dog brain atlas [14]. The corresponding ar巴asof the hippocampus were measured on the monitor at a magnificatio

was obtained after th巴transformationof the mean gray level using the formula: OD=log (256/mean gray level). The OD ofbackground was determined in unlabeled portions and the value subtracted for corr巴ction，yielding high OD values in the presence of preserved structures and low values after structuralloss using NIH lmage 1.59 software: A ratio ofthe relative optical density (ROD) was calibrated as %.

The result of the Western blot analysis was scanned， and the quantification of the Western blotting was done using Scion Image software (Scion Corp.， Frederick， MD，

U.S.Aふwhichwas used to count ROD: A ratio of the ROD was calibrated as %.

Statistical analysis: The data shown here represent the means :!:: SEM of experiments performed for each dentate area. Differences among the means were statistically ana-Iyz巴dby two-tailed Student t-test using SPSS program. Sta-tistical significance was considered at P<0.05.

RESULTS

GFAP immunoreactivity in the dentate gyrus: In the adult group， GFAP immunoreactive astrocytes (GFAP+ As) were detected in all layers of the dentate gyrus (Fig. 1 A). Many cell bodies of GFAP+ As were distributed in the polymor-phic layer. GFAP+ As in the dentate gyrus had thin pro-

cesses and thread-like morphology: Especially， the processes of GFAP+ As in the molecular layer were well ramified

In the aged group， although GFAP+ As were detected in alllayers of the dentate gyrus， the morphology of astrocytes was different from that in出eadult group (Fig. lB)， and the density of GFAP+ As in all layers was significantly lower than that in the adult group (Fig. 2A). Many GFAP+ As

were detected in the subgranular zone in the polymorphic layer and showed hypertrophied cytoplasm. Especially， GFAP+ processes in the molecular layer were very low in density compar巴dto those in the adult group (Fig. 2A).

GFAP immunoreactivity in the CAl-3 region: ln the CAI-3 r巴gionof the adult group， GFAP+ As were detected mainly in the strata oriens and radiatum; however， GFAP+ As were rarely detected in and near the stratum pyrarnidale (Fig. lC and IE).

In the aged group， GFAP+ As were detected in alllayer of the CAI-3 region (Fig. 10 and IF): The d巴nsityof GFAP+ structures in alllayers was significantly increased compared to the adult group (Fig. 2B). Especially， GFAP immunore-activity in the CA 1-3 regions was significantly increased in the stratum pyrarnidale compared to that in the adult group (Fig. 2B). In addition， the cell bodies and proc巴ssesin the aged groUP were hypertrophied compared to those in the adult group (Fig. 10 and lF).

GFAP levels in the hippocampus: ln this study， we found that changes in GFAP levels were somewhat different in the d巴ntategyrus and CA 1 region in adult and aged dogs. GFAP protein lev巴Isin the dentat巴gyrusof aged dogs were lower than that in the adult group. However， GFAP protein levels in the CA 1 region of aged dogs were higher than that

AGE-RELA TED CHANGES IN ASTROCYTES

Fig. 1. GFAP imrnunohistochemistry in the dentate gyrus (A and B)， CA 1 (C and D) and CA3 region (E and F) of adult (A， C and E) and aged (B， D and F) dogs. GFAP+ structures in the adult dentate

gyrus are abundant in all layers; however， GFAP+ structures in出eaged d巴ntategyrus decrease. In

the CA 1-3 region of the adult group， GFAP+ structures are mainly detected in strata oriens and radi-atum， however， GFAP immunoreactivity in the aged CA 1-3 region is markedly increased in alllay-ers. Note that GF AP+ astrocytes in the adult group have fine processes (inset in C)， however， in出e

ag巴dgroup， GFAP+ astrocytes show th巴hypeはrophyof cytoplasm (inset in D). GCL， granule cell layer; MoL， molecular layer， PoL， polymorphic layer; SO， stratum oriens; SP， stratum pyramidale;

SR， stratum radiatum. Bar = 100 μm (A-F)， 25μm (insets in C and D).

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• Adult group

MoL

• Adult group

ロAgedgroup

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in the adult group (Fig. 3). GFAP proteins in the whole hip-

pocampal homogenates were high in出eaged group com-

pared to those in由巴 adultgroup (Fig. 3).

DISCUSSION

The increase of GFAP immunoreactivity may be associ-

ated with the morphological activation of astrocytes as

astrogliosis under pathological condition. The astrogliosis

is characterized by the hypertrophy of cellular processes of

astrocytes and the up-regulation of GFAP in the CNS

ischernia， trauma or neurodegeneration [22]. In addition，

GFAP auto-antibodies are prevalent in the cerebrospinal

f1uids in dogs with various diseases [16， 24]. In this study， we used normal aged dogs and exarnined

that由巳rewere age-related differences in GFAP immunore-

activity and its protein levels in the dentate gyrus and CAト

3 region betwe巴nadult and aged dogs. The GFAP immu-

noreactivity and protein levels were decreased in the dentate

gyrus and markedly increased in the CAI-3 region in aged

dogs， however， GFAP+ As in both dentate gyrus and CAI-3

region were hypertrophied.

Fig.2. Relative optical density (ROD) as % GFAP immunoreac-tivity in the dentate gyrus (A) and CAI-3 region (B) of adult and aged dogs (n=5 per group; * P<O.05， significantly different from the adult group). Data are express巴das the means :t SEM

There are some contradictory reports on the age-related

changes of GFAP+ As in rodent brains. A few studies

reported that the significant age-related changes of astro-

cytes were not exarnined in the mouse hippocampus [15， 19]; however， many studies showed the age-related eleva-

tions of GFAP rnRNA in the rat hippocampus and striatum

[20]， protein levels in the mouse striatum [13， 21] and the number of GFAP+ As in various brain regions (i.e.， hippoc-

ampus， cerebral cortex and striatum) [1，2，7，9，23，25，27] However， these studies did not demonstrate the changes of

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Dentate gyrus

CAl・3region

Whole hippocampus

Adult Aged

* • Adult group

ロAgedgroup

*

Dentate gyrus CAl-3 region Wbole Hippocampus

Fig.3. The top panel shows Westem blot analyses for GFAP in the dentate gyrus， CAI-3 region and whole hippocampus of the adult and aged group. The bottom panel shows relative optical density (ROD) as % of immunoblot bands (* P<O.05， significantly different from the adult group). Data are expressed as the means土SEM

AGE-RELA TED CHANGES IN ASTROCYTES 969

GFAP immunoreactivity in the subregions of the dog hip-

pocampus.

ln this study， we found that GFAP+ structures were mark-

edly decreased in the dentate gyrus in aged dogs. There is

no study on decrease in GFAP+ structures in the dentate gyrus

in aged animals. It has been reported that bouton density was significantly decreased within 200μm of compact s-amyloid

plaques in th巴outermolecular layer of the dentate gyrus at

15-18 months of age in Tg 2576 mice [5]. Therefore， we

suggest that the decrease of GFAP+ structures in the aged

dentate gyrus may be related to the decrease of synapses.

On the other hand， it has been reported that moderate

astrocytic gliosis was observed in the aged dog hippocampus

[2， 25]. ln addition， electron microscopy demonstrated the increased number of pro臼esof degenerative neural compo-

nents in the vicinity of hypertrophic astrocytes in the cere-

bral cortex of the aged dogs [25]. However， these studies did

not demonstrate the chang巴sof GFAP immunoreactivity in

the subregions of the dog hippocampus: They studied on the

overall changes of GFAP+ As in the whole dog hippocam-

pus. ln conclusion， our present study shows that， in the aged dog， GFAP immunoreactivity is markedly increased in the

CAl-3 regions， but slightly decreased in the dentate gyrus.

The regional differences in GFAP+ As may be associated

with neuronal vulnerability in these subregions.

ACKNOWLEDGEMENTS. The authors would like to

thank Mr. Seung Uk Lee and Ms. Hyun Sook Kim for their

technical help in this study. This work was supported by the

Korea Research Foundation Grant funded by the Korean

Govemment (MOEHRD) (KRF-2007-313-E00048)

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